Since the regulatory role of adenylate cyclase has been recognized as important to the development of Dictyostelium discoideum, quantitation of the amount of cAMP formed by this enzyme has been of interest. Techniques used to detect the cAMP formed include those using (32P) ATP as substrate or a radioimmunoassay (RIA). We have found that formycin 5' -triphosphate (FoTP), a fluorescent analog of ATP, is a substrate for the adenylate cyclase from D. discoideum and developed a novel fluorometric assay for detection of activity of this enzyme. This procedure utilizes high pressure liquid chromatography (HPLC) and reverse phase chromatography to separate the reaction products. Calibration of the HPLC and standardization of other procedures such as the RIA is carried out with authentic cyclic FoMP and procedures are described to carry out the synthesis of this analog as well as FoTP and FoMP. A number of methods will be used to validate the formation of cFoMP including the RIA, susceptibility to degradation by cAMP phosphodiesterase and the formation of (32P) cFoMP from alpha (32P) FoTP. Methods are described for the formation of alpha (32P) FoTP enzymatically using a newly discovered activity from D. discoideum namely adenosine kinase and commercial adenylate cyclase. This method offers a number of distinct advantages over those presently in use including the fact that no radioactive materials are required and is therefore environmentally safe, and it is able to be completely automated. Also, experiments are described in which I plan to use thioanalogs of ATP including ATP beta S (A and B) as substrates for the adenylate cyclase activity. These experiments will include a study of possible stereospecificity and the metal-dependence of the stereospecificity. I propose similar experiments with the ATP pyrophosphohydrolase to probe further the role of this activity in the regulation of cyclase activity. Experiments on the developmental regulation of the ATP pyrophosphohydrolase activity are also described.